1. Field of the Invention
The present invention concerns the field of electrochemical devices for detection and measurement purposes and more specifically an enzyme emulsion for use in an implantable miniature polarographic glucose sensor.
2. Description of Related Art
There is currently a considerable need for a glucose sensor that can be readily implanted into a human where it will function for a prolonged time period. The primary impetus for such a device is diabetes, a potentially devastating complex disorder of glucose metabolism, currently controllable through insulin injections, is increasing worldwide. In the United States it is estimated that over ten million persons have diabetes. The monetary cost to society is in the many billions of dollars reflecting treatment expense and loss of productivity while the human cost in impaired function, progression to blindness, limb amputations, kidney failure and heart and vascular disease is immeasurable.
It has been known for well over seventy years that this disease primarily results from inadequate secretion of the hormone insulin by the islet or Beta cells of the pancreas. When uncontrolled, this disease often leads to serious metabolic imbalancesxe2x80x94elevated glucose levels lead to ketosis and to damaging alterations in blood pH while inadequate glucose levels lead to lethargy and coma. Diet and daily injections of insulin are now used in an attempt to control life-threatening swings in blood glucose. It is now well established that the damage is caused by excessive glucose and not directly by lack of insulin. Glucose combines with hundreds of proteins essential for normal metabolism and in that way damages the cellular machinery of the body.
Control of diabetes by insulin injection generally results in much wider swings in blood glucose level than are common in a normal individual. Occasional insulin injections (up to several per day) are unable to duplicate the strict control of blood glucose afforded by a properly functioning pancreas which continually meters out just enough insulin to maintain a stable and relatively normal blood glucose level. Extremes in blood glucose level need be avoided. Yet despite avoiding extremes in blood glucose level insulin-dependent diabetics suffer a host of other maladies, mentioned above, that decrease both the quality and length of life. Diabetics experience frequent vascular disease that often results in amputation of limbs as impaired circulation prevents adequate blood flow. Abnormal vascular growth within the eye may result in intraocular bleeding and retinal damage with progressive loss of vision. Nerve degeneration may lead to loss of sensation and other related problems.
To control the blood level of glucose by injection of insulin requires the analysis of six to eight samples of blood each day. This is usually performed by puncturing the finger tip with a small lancet and analyzing blood glucose level with a photometric xe2x80x9chome glucose monitor.xe2x80x9d This is, of course, not a pleasant experience and requires considerable skill as well as motivation. As home glucose tests have became common, more and more data have became available demonstrating the relatively poor control of blood glucose afforded by periodic insulin injections. At the same time, a growing number of clinical studies demonstrated that strict control of blood glucose reduces many if not all of the diabetes-related diseases mentioned above. Many scientists and physicians now believe that greatly improved blood glucose control can largely eliminate the mortality and morbidity associated with diabetes.
The ultimate goal of diabetes treatment is a replacement for the patient""s non-functioning pancreatic islet cells. Some scientists are seeking ways to transplant functioning islet cells into diabetic patients to provide a naturally controlled source of insulin. Other scientists are working on automatic insulin injection systems that deliver exogenously supplied insulin as needed to maintain precise blood glucose control. Most probably both of these xe2x80x9ccuresxe2x80x9d will be needed. Although transplanted islet cells would seem to be the optimal solution, at this time anti-rejection drugs required for transplants have almost as many negative side effects as diabetes itself. In any case, a self-regulating artificial insulin source is needed to limit the damage caused by diabetes until islet transplantation is perfected. Even when transplantation is widely available, a self regulating insulin source will be needed for patient maintenance prior to transplantation, and, perhaps, for some post-transplantation support.
Many types of regulated injection systems, both implantable and external, are already available. The key problem continues to be the requirement for an accurate glucose sensor to control these injection systems. The need to continually monitor glucose levels to permit a constantly metered dispensing of insulin generally eliminates methods relying on blood samples. It is clear that an implantable glucose sensor that measures in vivo glucose levels is the real answer.
Previous to modem instrumentation the analysis for blood glucose required a venipuncture with the collection of several milliliters of blood, precipitation, filtration, treatment with a colorimetric glucose reagent and spectrophotometric determination of glucose. The invention of the first xe2x80x9cenzyme electrodexe2x80x9d and glucose sensor by the present inventor in the 1960""s led to the production of the first commercially successful blood glucose analyzer. The Clark glucose sensor consisted of a platinum anode, a layer of glucose oxygen oxioreductase (glucose oxidase) and a cellophane or cellulose acetate membrane. A silver xe2x80x9creferencexe2x80x9d electrode was also incorporated into the sensor. Only 0.01 ml of blood was required and the final analysis was complete in about one minute. Since then literally billions of blood samples have been analyzed by this type of instrument.
The inventor""s polarographic glucose method just mentioned is explained in U.S. Pat. No. 3,539,455. The chemical reaction most commonly used by such enzyme-coupled polarographic glucose sensors is glucose oxidase mediated catalytic oxidation of glucose by atmospheric oxygen to produce gluconolactone and hydrogen peroxide (equation 1):
C6H12O6+O2+H2Oxe2x86x92C6H12O7+H2O2xe2x80x83xe2x80x83(1)
In the presence of excess oxygen, the quantity of hydrogen peroxide produced will be a direct measure of the glucose concentration. The hydrogen peroxide is measured by being reoxidized by an electrode (anode) maintained at an appropriate positive potential (equation 2):
H2O2xe2x88x922exe2x88x92xe2x86x92O2+2H+xe2x80x83xe2x80x83(2)
The glucose detection process, then, is dependent upon the measurement of electrons removed from hydrogen peroxide in equation (2). The electrode is normally formed from a noble metal such as gold or platinum. The latter preferred metal although carbon, pyrolytic or glassy, graphite and other electrically conducting materials are sometimes used.
As is well known to those of ordinary skill in the art, other specific hydrogen peroxide producing oxidase enzymes can be used to produce sensors for other substances such as cholesterol (cholesterol oxidase), amino acids (amino acid oxidase), alcohol (alcohol oxidase), lactic acid (lactate oxidase), and galactose (galactose oxidase), to name only a few.
The success of this kind of enzyme-based sensor suggested to many that a similar sensor might be implanted with a simple power source and a means for transmitting the glucose data to the outside of the body. Such a continuously reading device would not only eliminate the pain of repeatedly puncturing the finger but would also supply a constant reading of the glucose level. It is known that the glucose level in many locations in the body closely mirror the blood glucose level. Numerous attempts have been made to make such a device available to diabetics. However, experimental devices did not function a sufficiently long period of time. These failures of implanted glucose sensors were ascribed to diverse problems, many of which appeared to be without solution. For example, some believed that the hydrogen peroxide (and free radicals) generated by the oxidase reaction caused denaturation and inactivation of the oxidase enzyme. Another, more common, explanation was that the glucose sensor was xe2x80x9cnot compatiblexe2x80x9d with the human body or that the surface of the measuring tip of the electrode became coated with layers of scar-like tissue which not only impeded the diffusion of glucose but jeopardized or destroyed nearby capillaries. Others held that the platinum electrode surface became xe2x80x9cpoisonedxe2x80x9d by body fluids. However, extensive studies of implanted platinum electrodes conducted in the inventor""s laboratory over the last forty years have completely exploded the myth of the xe2x80x9cpoisonedxe2x80x9d platinum surface. Some implanted platinum electrodes have remained functional for up to six years.
It is a goal of this invention to similarly deal with other impediments to successful implantable glucose sensors. Glucose is extremely soluble in biological fluids whereas oxygen is poorly soluble in these same fluids and must be carried by specialized biomolecules such as hemoglobin. Many tissues of the human body have an oxygen tension equivalent to between about 2-5% oxygen in nitrogen or lower. As a result, there may be a ratio of glucose to oxygen sometimes as high as 100 to 1 in subcutaneous interstitial and peritoneal fluids. This means that at the electrode surface there may be only 1% of the oxygen required for glucose oxidase to quantitatively oxidize the available glucose for measurement purposes.
Furthermore, the glucose oxidase in a glucose sensor must be protected from proteases and other macromolecules which might destroy or inhibit the glucose oxidase, from enzymes such as catalase which destroy hydrogen peroxide (catalase, dehydrogenases, etc.), from microbes which digest the enzymes and from soluble compounds, such as ascorbate and acetoaminophen, which interfere with the either the enzymatic or electrochemical reactions. This protection can be achieved by separating the glucose oxidase from biological fluids by a semipermeable membrane. The best known membranes that are capable of selectively excluding proteins such as catalase while allowing the entry of glucose are so-called dialysis membranes. These membranes are generally hydrophilic membranes containing xe2x80x9cporesxe2x80x9d that readily admit neutral molecules with molecular weights below about 5,000 Daltons. Common examples of these membranes are prepared from various regenerated celluloses such as cellophane, Spectrapore(copyright) or Cuprophan(copyright) (brands of regenerated cellulose), cellulose esters, and membranes of polycarbonate or polysulfone.
While such semipermeable membranes do a good job of excluding undesirable proteins as well as retaining the essential glucose oxidase, they also impede oxygen diffusion. Some membranes, however, such as those of polytetrafluoroethylene (Teflon(copyright) brand of perfluorocarbon resin) or of silicone rubber are permeable to oxygen, but these membranes are virtually impermeable to glucose, and hence, cannot be used to protect an oxygen requiring glucose sensor. U.S. Pat. No. 5,322,063 to Allen et al. reports a new type of polyurethane membrane said to allows some glucose permeability while favoring oxygen permeability.
Because of a superabundance of glucose and a shortage of oxygen, an implanted glucose sensor will tend to be oxygen limited and, thus, effectively measure oxygen instead of, or together with, glucose. That is, under ideal conditions when the glucose concentration is low, oxygen would be adequate so that an increase in glucose concentration would result in a concomitant and proportional increase in hydrogen peroxide and, therefore, measured current at the electrode. However, as the concentration of glucose increases, oxygen ultimately becomes insufficient causing the measured current to plateau regardless of glucose concentration. Above this plateau changes in the current reflect changes in oxygen tension (concentration) rather than in glucose concentration.
Many workers have failed to take into account the high glucose to oxygen ratio of human tissues. There are at least two ways to solve this problem: one can attempt to reduce the concentration of glucose that reaches the glucose sensor and/or one can attempt to increase the amount of oxygen available at the glucose sensor. The level of glucose can be reduced either by providing a permeability barrier to glucose or by providing additional, non-peroxide generating enzyme systems, such as dehydrogenases, besides glucose oxidase, to consume excess glucose. The polyurethane membrane mentioned above is an example of glucose restriction.
The second approach involves an attempt to increase the level of available oxygen or to maximize the availability of oxygen to the oxygen-requiring enzymes. The present inventor has previously disclosed methods for increasing the oxygen level in U.S. Pat. Nos. 4,680,268 and 4,721,677, which are hereby incorporated by reference. These patents teach the use of an oxygen-collecting chamber made of an oxygen permeable material such as silicone rubber. This chamber is separated from the oxygen-requiring enzyme by an oxygen permeable membrane. The chamber collects oxygen and delivers it by diffusion to the enzyme mixture near the measuring electrode. These patents also disclose filling the oxygen-collecting chamber with an oxygen-dissolving compound such as a perfluorocarbon liquid for speeding diffusion of oxygen to the oxygen-requiring enzyme. Alternatively, an emulsion of a perfluorocarbon liquid and the enzyme solution could be used to fill the chamber with the device configured so that the emulsion flows slowly onto the electrode, supplying oxygen and replenishing the enzyme. A recent publication (Wang and Lu, J. American Chem. Soc. 120:1048-50(1998)) adopts this liquid emulsion strategy but add graphite or carbon powder so the emulsion also functions directly as an electrode. This could cause difficulties with an implantable electrode since macrophages might react to the carbon powder.
Experiments in the inventor""s laboratory have also showed that the current output from chronically implanted sensors can be used to control ascorbic acid levels in the brain using a feedback loop to an ascorbate pump and connected indwelling catheter. Further, it has been shown that polarographic anodes can be used quantitatively to measure blood flow in the vicinal capillary beds. It has been found from thousands of hours or continuous recording, that the oxygen available to the surface of an implanted oxygen sensor, and therefore to the glucose sensor described herein, is not steady but waxes and wanes in waves of six to eight cycles per minute. In some cases the amplitude of this variation can be plus or minus 30%. Buffering such fluctuation in available oxygen would be highly desirable in an oxygen-requiring glucose sensor since the functioning of such sensors depends upon oxygen.
In glucose sensor longevity experiments in the inventor""s laboratory using sensors implanted in the peritoneal space of mice it was found that some sensors retained full activity for over 400 days. Over time the activity of most sensors gradually declined. These data demonstrated that adequate longevity could be achieved but that some factor, perhaps mechanical, frequently caused loss of activity. The invention disclosed herein is designed to avoid microbial degradation of the enzyme as well as to resist attacks by free radicals, proteases and the host""s immune system.
The present inventor has discovered that xe2x80x9coxygen sensitivityxe2x80x9d of enzyme-based polarographic electrodes can be significantly reduced or eliminated by providing an oxygen-reservoir in intimate contact with the oxidative enzyme. This is achieved by making a stabilized emulsion of the enzyme and a compound in which oxygen is extremely soluble. For example, an aqueous glucose oxidase solution can be emulsified with a perfluorocarbon liquid and the resulting emulsion stabilized by chemically crosslinking the mixture to form a gel. Thin layers of the emulsion ideal for placing into contact with a noble metal electrode can be fabricated by spreading a layer of the emulsion prior to crosslinking. Additional carrier proteins such as albumin can be added to the oxidase prior to crosslinking to protect enzymatic activity from the crosslinking reagent.